1. Field of the Invention
The present invention relates to the technology of a confocal scanning microscope using a confocal effect, and also to the three-dimensional measurement technology for obtaining the surface data of a sample using an image of a color-captured sample (including a measured object) through a confocal optical system. It also relates to the technology of selecting as the measuring range of the sample a predetermined area of a three-dimensional confocal microscope having color information depending on the sample obtained by the confocal scanning microscope.
2. Description of the Related Art
Generally, two types of confocal scanning microscopes, that is, a confocal disk scanning microscope and a confocal laser scanning microscope, are well known. The confocal disk scanning microscope is not only higher in horizontal resolution than a common microscope, but also is higher in sectioning effect in the optical axial direction (hereinafter also referred to as a “Z direction”) of a sample. Based on these characteristics, it can be used with an image processing apparatus to generate the sample as a three-dimensional image.
FIG. 1 shows the configuration of the conventional confocal disk scanning microscope.
In FIG. 1, the illuminating light is emitted from a light source 1 enters a half mirror 3 through a collimator lens 2, reflected by the mirror, and illuminates a rotary disk 4. The rotary disk 4 can be a Nipkow disk for which a plurality of pinholes are provided in a spiral manner, a disk having a slit pattern, etc. In this example, a Nipkow disk is used, and the rotary disk 4 is attached to a rotation axis 5a of a motor 5, and rotates at a predetermined rotation speed. Therefore, the illuminating light irradiating the rotary disk 4 transmits through a plurality of pinholes formed in the rotary disk 4, and is formed as an image on a sample 7 by an objective lens 6.
The reflected light from the sample 7 passes through the objective lens 6 and the pinholes of the rotary disk 4, and transmits through the half mirror 3, and is formed as an image on a capture unit 9 by a converging lens 8. The capture unit 9 captures the reflected light from the sample 7, and outputs the brightness signal to a computer 10.
The computer 10 captures the brightness signal output from the capture unit 9, stores it, performs image processing, obtains predetermined image data, displays it on a monitor 11, simultaneously outputs a drive signal to a Z drive unit 12, moves all or a part of the optical system and the sample relatively in the optical axial direction, and changes the focal-plane position (focal position) of the sample. In this example, only the objective lens 6 is moved, but, for example, a stage loaded with the sample 7 can be moved in the optical axial direction. Then, the focal-plane position which is position information in the Z direction is associated with the above-mentioned image data and stored in the computer 10.
With the above-mentioned configuration, the confocal disk scanning microscope changes the amount of incident light to the capture unit 9 by moving the objective lens 6, and marks the maximum amount of incident light (brightness) when focus is achieved on the surface of the sample. Since each pixel of the image pickup device in the capture unit 9 outputs a brightness signal corresponding to the amount of light from each position of the sample 7, the three-dimensional shape of the sample 7 can be obtained by obtaining the position in the z direction of the highest brightness of each pixel, and an image can be generated only by the maximum brightness value of each pixel, thereby generating an image having a large depth of focus with focus achieved on the entire surface of the sample 7.
FIG. 2 shows the configuration of the conventional confocal laser scanning microscope.
In FIG. 2, the light emitted from a light source 13 transmits through a beam splitter 14, and enters a two-dimensional scanning mechanism 15. The two-dimensional scanning mechanism 15 includes a first optical scanner (X scanner) 15a and a second optical scanner (Y scanner) 15b, and each of the optical scanners 15a and 15b performs optical scanning using optical flux in a two-dimensional manner. By leading the optical flux to an objective lens 16, the optical flux enters the objective lens 16, converges by the objective lens 16, and scans the surface of a sample 17 in a two-dimensional manner. The light from the surface of the sample 17 passes through the objective lens 16 again, reaches the beam splitter 14 through the two-dimensional scanning mechanism 15, is reflected by the beam splitter 14 and converged on pinholes 19 by an imaging lens 18. Since the pinholes 19 are located in a position optically conjugate with the converging position, the light from the sample 17 is converged on the pinholes 19 and passes through the pinholes 19 when the sample 17 is in the converging position of the objective lens 16, but is not converged on the pinholes 19 and does not pass through the pinhole 19 when the sample 17 is not in the converging position of the objective lens 16. Therefore, the light from the sample 17 cannot pass through the pinholes 19 on the points other than the converging point of the objective lens, and only the light passing through the pinholes 19 is detected by a photodetector 20. The sample 17 is placed on a sample mount 22 corresponding to the X and Y stages, and can be moved by a Z stage 23 in the optical axial direction. The two-dimensional scanning mechanism 15, the Z stage 23, and the photodetector 20 are controlled by a computer 21.
With the above-mentioned configuration, if the two-dimensional scanning mechanism 15 performs two-dimensional scanning and imaging the output of the photodetector 20 in synchronization with the two-dimensional scanning mechanism 15, then the imaging operation is performed only for the specific height of the sample 17, thereby obtaining an optically sliced image (confocal microscopic image) of the sample 17. Furthermore, by discretely moving the sample 17 in the optical axial direction (Z direction) by the Z stage 23, operating the two-dimensional scanning mechanism 15 in each position to obtain a confocal microscopic image, and detecting the position of the Z stage 23 in which the output (brightness value) of the photodetector 20 is the highest at each point of the sample surface, the surface form (surface form information, height information) of the sample 17 can be obtained, and a three-dimensional image (three-dimensional confocal microscopic image) can be obtained depending on the surface form. Furthermore, by forming an image only with the output of the photodetector 20 which indicates the highest value at each point of the sample surface, an image having a large depth of focus with focus achieved on the entire surface of the sample 17.
To obtain a clear and high precision image with the above-mentioned confocal scanning microscope, it is necessary to have a steep curve (hereinafter referred to also as an “I-Z curve”) indicating the relationship between the brightness value and the position in the Z direction.
FIGS. 3 and 4 show an example of an I-Z curve obtained by the above-mentioned confocal disk scanning microscope.
FIG. 3 shows an I-Z curve of the wavelength of green (G) as a characteristic in a narrow wavelength band. In FIG. 3, the peak of the brightness value indicating the highest brightness can be clearly discriminated. On the other hand, when the wavelength band of the illuminating light is not limited in a normal optical system, an obtained I-Z curve indicates a moderate peak mainly by the influence of the chromatic aberration generated by the objective lens 6. FIG. 4 shows the state in which there are different positions indicating the highest brightness of each wavelength of red (R), green (G), and blue (B) by the chromatic aberration of the lens, and the state in which the I-Z curve (white) of white light obtained by combining them has a moderate peak.
Therefore, using a narrow wavelength band by inserting a wavelength filter at the light source or in the capture unit, a steep I-Z curve can be obtained. In this case, the formed three-dimensional image is very precise.
FIG. 5 shows an example of another I-Z curve obtained with the confocal scanning microscope, and shows an example of an I-Z curve of each color component indicating an example of the relationship between the amount of displacement and the brightness value of a specific pixel of an image obtained by color-capturing the light of plural wavelengths from the sample surface through the confocal optical system while relatively displacing the focal-plane of the confocal optical system and the sample. The horizontal axis indicates the amount of displacement Z between the focal-plane of the confocal optical system and the sample, and the vertical axis indicates the brightness value I.
As shown in FIG. 5, the I-Z curve of each color component of R (red), G (green), and B (blue) color-captured by the color-capture unit indicates a different amplitude in brightness and peak position due to the optical characteristic on the surface of the sample, the wavelength dependence, and the aberration of the optical system. Each I-Z curve indicates a unimodal peak. The peak position matches the amount of displacement when focus is achieved on a small area of the sample surface. Therefore, as with other pixels within the vision, the peak position of the I-Z curve as shown in FIG. 5 can be detected, thereby obtaining the amount of displacement corresponding to the height of the surface in each position of the sample, and obtaining the surface form of the sample.
Thus, when the obtained surface form of a sample is observed, the surface form can be displayed in a three-dimensional array for easier visual recognition.
FIGS. 6, 7, and 8 show an example of the three-dimensional display method.
In FIGS. 6, 7, and 8, the method is to obtain a three-dimensional display of a sample colored as shown in FIG. 8 by coloring the three-dimensional display of the sample as shown in FIG. 7 using an observed image of the sample as shown in FIG. 6. As an observed image of a sample used in this example is an image of the sample surface with focus achieved in the entire position within the vision. That is, about the image, the color information in the peak position of the I-Z curve obtained in each position within the vision to obtain the surface form is the color information about the corresponding position within the vision.
Recently, as shown in FIGS. 6 through 8, there are some technologies of coloring an obtained three-dimensional image proposed to easily understand the correspondence with a sample. For example, Japanese Patent Application Laid-open No. 2001-82935 discloses the confocal color microscope for obtaining a three-dimensional color image (three-dimensional confocal color microscopic image) by combining a three-dimensional image depending on the surface form (surface form information) of the sample obtained by a confocal optical system with a color image according to the color information about a sample obtained by a nonconfocal optical system.